Method and assay for detection of residues

ABSTRACT

Embodiments described herein include methods and assays for detecting an analyte in a sample using a plurality of control zone capture agents. Some embodiments include detection of multiple analytes in a sample utilizing a plurality of analyte binders and a control zone containing multiple control zone capture agents. In some embodiments, the multiple control zone capture agents capture a plurality of binders within one control zone. Test results are determined by comparison of the control zone signal to a test zone signal.

REFERENCE TO PRIOR APPLICATION

This application is based on and claims priority from U.S. ProvisionalPatent Application Ser. No. 60/527,934, filed Dec. 8, 2003, which isincorporated by reference.

BACKGROUND

Antibiotic residues in foods are a major food safety concern. Healthissues include bacterial resistance to drugs and allergic reactions. Toavoid the impact of such health issues, food is tested worldwide forantibiotics and other contaminants. One type of test method uses what iscommonly known as a lateral flow test strip.

Lateral-flow test strips for detecting one or more analytes in a fluidsample may include a capture agent immobilized within a region of thetest sometimes referred to as a detection zone. Detection zones caninclude test zones and control zones. A typical capture agent hasbinding affinity for a substance that may be in the mobile phase of thetest strip.

Lateral-flow tests in which the binding of a substance from a mobilephase to a capture agent generates a visible signal, that can beinterpreted visually or using a reader, such as a spectrophotomer, arewell known in the art. Examples of such devices are described in U.S.Pat. No. 5,985,675, issued Nov. 16, 1999; and U.S. Pat. No. 6,319,466,issued Nov. 20, 2001, and U.S. patent application Ser. No. 10,289,089,filed Nov. 6, 2002 (based on U.S. Provisional Application 60/332,877,filed Nov. 6, 2001) all of which are incorporated herein by reference.

Lateral-flow tests are widely used in the food products industry. Oneapplication is in testing milk. The United States Food and DrugAdministration (FDA) requires that bulk milk tankers be tested forunsafe levels of beta-lactam antibiotics. The sensitivity requiredpursuant to Appendix N of the Pasteurized Milk Ordinance is: amoxicillin10 parts per billion (“ppb”), ampicillin 10 ppb, penicillin G 5 ppb,ceftiofur 50 ppb, cephapirin 20 ppb, and cloxacillin 10 ppb (the “safelevels”). The FDA also requires that certain milk tests be automatically(electronically) analyzed and recorded. It is desirable, therefore, toprovide milk-testing personnel with a user-friendly test that can beanalyzed with or without a reader and can detect multiple beta-lactams,including the six beta-lactams listed above (“the target beta-lactams”),when present at or above the safe level.

SUMMARY

Disclosed herein is a method and apparatus, such as a lateral flow teststrip type test, for detection of a contaminant in a sample. Antigens,haptens and their antibodies, hormones, vitamins, drugs, metabolites andtheir receptors and binding materials, fungicides, herbicides,pesticides, plant, animal and microbial toxins, may be determinableusing the present methods and apparatuses. Other analytes that may bedeterminable by the disclosed methods and apparatuses includeantibiotics, such as beta-lactams, cephalosporins, erythromycin,sulfonamides, tetracyclines, nitrofurans, quinolones, vancomycin,gentamicin, amikacin, chloramphenicol, streptomycin and tobramycin andtoxins, such as mycotoxins, such as aflatoxin and vomitoxin and drugs ofabuse, such as opioids and the like, as well as the metabolites thereof.

The test can include one or multiple binders each with binding affinityfor one or more analytes. The test can also include a test zone captureagent and one or more control zone capture agents with binding affinityfor the binders. In an example, two control zone capture agents are usedeach with affinity to a different binder. In another example, twocontrol zone capture agents are used and the two control zone captureagents also have binding affinity to each other.

An example described herein is a test for detection of beta-lactamantibiotics, including test strips sensitive to penicillin G,amoxicillin, ampicillin, ceftiofur, cephapirin and cloxacillin withsensitivity to each at or below safe level. Such a test can include abinder for beta-lactam antibiotics, for example a beta-lactam bindingprotein derived from Geobacillus stearothermophilus (sometimes referredto as Bacillus stearothermophilus) (“B.st.”) with affinity to multiplebeta-lactams including the target beta-lactams (“the BL binder”).Generally binders with affinity to multiple drugs are hereinafterreferred to as “multianalyte binders”.

The test can also include a binder with greater specificity for aparticular analyte as compared to a multianalyte binder, hereinafterreferred to as a “specific binder”, for example an antibody to anantibiotic such as cloxacillin (“cloxacillin binder”) to which themultianalyte binder, such as the BL binder, may not have adequatesensitivity. In such a test, a test zone can contain a capture agent forthe binder, for example representative antibiotics. When two binders areused, such as a multianalyte binder and a specific binder, a detectionzone can include capture agents for both, for example capture agentsimmobilized in separate test zones. Immobilization can be through use ofa carrier protein, such as BSA.

The test can also utilize two or more binders having sensitivity forunrelated analytes such as different families of antibiotics or toxins.

A control zone can be used for comparison to the one or more test zonesor as a signal that the test functioned properly and is complete. Acontrol zone can also include a capture agent. In an example, one of thecontrol zone capture agents includes an antibody binding protein such asprotein A, protein G or protein AG or recombinant forms of the same. Inanother example, one of the control zone capture agents includes anantibody, for example an antibody to a multianalyte binder immobilizedon the control zone prior to testing. It is also possible that anantibody to a multianalyte beta lactam binder is not immobilized in thecontrol zone and is instead combined with a beta lactam binder prior totesting and flows to the control zone for capture. In such an example,there is a single capture agent immobilized on the control zone prior totesting which can be an antibody binding protein.

When the control zone capture agents include an antibody bindingprotein, and an antibody, the capture agents may have affinity to eachother, and, therefore, if combined, may have binding to each other. Thecontrol zone capture agents need not, however, have affinity to eachother. For example, the control zone capture agents can include avariety of antibodies, receptors, binding proteins and the like, eachwith affinity to at least one of the analyte binders. Generally,however, when multiple test zones are employed, it is preferable to havea control zone be used for comparison to more than one test zone. Inthat way, one control zone can be used to compare to more than one testzone, thereby simplifying test result interpretation. For example, ifthere are two test zones one control zone can be used, or if there arefour test zones then two control zones can be used.

In another aspect, in which one test zone and one control zone are usedto capture one labeled binder, the control zone includes multiplecapture agents, each with different affinity to a binder, such as eachwith affinity to different parts of the binder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a side view of an embodiment having two test zones and acontrol zone.

FIG. 2 is a schematic view of the arrangement of test components on thetest strip prior to application of sample.

FIG. 3 is a schematic view of the test components after application ofsample. The movement of fluid and formation of complexes is shown in anexample of a test that is negative for both cloxacillin specifically andbeta-lactams.

FIG. 4 is a schematic view of the test components after application ofsample. Movement of fluid and formation of complexes is shown in anexample of a test that is positive for both cloxacillin specifically andbeta-lactams.

FIG. 5 is a schematic view of the test components after application ofsample. Movement of fluid and formation of complexes is shown in anexample of a test that is negative for cloxacillin specifically andpositive for beta-lactams.

FIG. 6 is a schematic view of the test components after application ofsample. Movement of fluid and formation of complexes is shown in anexample of a test that is positive for cloxacillin specifically andnegative for beta-lactams.

DETAILED DESCRIPTION

An analyte or chemical residue test device and method for detecting ananalyte in a sample is described herein. Often a substance such as milkhas one or more analytes that need to be detected. To detect an analytea binder for the analyte can be employed. The binder can be a bindingprotein such as an enzyme, antibody, receptor or other substance capableof binding to the analyte to form an analyte-binder complex. The binder,or analyte-binder complex, can be detected through various methodsincluding labeling the binder, and, therefore, the resulting complex,with a visible label, such as a gold particle, and capturing the labeledcomplex with a capture agent.

In embodiments utilizing a lateral flow test strip, the strip caninclude a solid support, such as nitrocellulose with sufficient poresize to allow liquid to flow along the membrane, and a separate regionfor applying a fluid sample (“sample pad”). The test strip can includeone or a plurality of binders selected for their binding affinity forthe analytes to be detected. Binders can be located on the sample pad orelsewhere on the test strip.

In an example of using the test strip, fluid is applied to a sample pad.The sample fluid solubilizes the binders and migrates through the teststrip by the forces of lateral capillary flow. The binders bind toanalyte in the sample. The fluid flows to a detection zone. Thedetection zone can include one or more test zones and one or morecontrol zones, each containing capture agents.

In an example, a test zone capture agent can be, for example, ananalyte, representative analyte, or analyte analogue. The capture agentat some point must be immobilized to the strip so that it is eitherremoved from sample flow or is not solubilized by sample fluid flow.Immobilization on the strip, so that the capture agent is notsolubilized by fluid flow, can be accomplished using a carrier proteinsuch as bovine serum albumin (BSA), or other carrier protein well knownin the art, for example ovalbumin (OVA) or keyhole limpet hemocyanin(KLH).

Each test zone capture agent captures all or a portion of the binder,from what is known as the mobile phase, which is not already bound withsample analyte. A binder that is bound by analyte from the sample tendsnot to be captured at the test zone. Binders that are not captured atthe test zone can be captured in the control zone or flow through to adisposal pad.

In an embodiment, a specific binder is a cloxacillin binder and amultianalyte binder is a BL binder. Both the cloxacillin binder and theBL binder can be detectably labeled, for example using gold particles.The labeled cloxacillin binder and labeled beta-lactam binder can becombined in a solution and applied, for example, by spraying, within orproximate to a pretreated POREX® (POREX is a registered trademark ofPorex Technologies Corp, Georgia USA) sample pad in contact with anitrocellulose membrane. The binders can also be combined with thesample in a container, such as a test tube, and added to the test stripwith the sample. When exposed to a sample such as fluid milk, thecloxacillin binder binds to cloxacillin in the milk and the BL binderbinds to beta-lactams (including to some extent cloxacillin) in the milkto form complexes. Lateral capillary flow carries the complexes, and anyuncomplexed labeled binders, to the test zone area of the strip.

In an embodiment using two binders, multiple test zones can be employedto capture the binders in separate zones. In an example in whichcloxacillin binder and BL binder are used, the first test zone captureagent can include immobilized cloxacillin and the second test zonecapture agent can include an immobilized different beta-lactam, such asceforanide. In an embodiment in which the positions are reversed, thefirst test zone capture agent can include a representative beta-lactamand the second test zone capture agent can include cloxacillin. In thetest zones, the capture agents can capture the binders that have notbeen previously bound by sample analyte. Such attachment at the testzone can generate a visible signal when a detectable label, such as goldor other label well known in the art, is used. Other particles that maybe useful include, but are not limited to, colloidal sulphur particles;colloidal selenium particles; colloidal barium sulfate particles;colloidal iron sulfate particles; metal iodate particles; silver halideparticles; silica particles; colloidal metal (hydrous) oxide particles;colloidal metal sulfide particles; colloidal lead selenide particles;colloidal cadmium selenide particles; colloidal metal phosphateparticles, colloidal metal ferrite particles, any of the above-mentionedcolloidal particles coated with an organic or inorganic layer; proteinor peptide molecules; liposomes; or organic polymer latex particles,such as polystyrene latex beads. Other labels may also be usefulincluding, but not limited to, luminescent labels; fluorescent labels;or chemical labels, such as electroactive agents (e.g., ferrocyanide);enzymes; radioactive labels; or radiofrequency labels.

In an embodiment, two control zone capture agents are employed withinthe same control zone. The capture agents can each capture a differentbinder. In an embodiment in which a cloxacillin binder and a BL binderare both employed, protein A can be one control zone capture agent andan antibody to beta-lactam binder (“anti-BL binder”) can be the othercapture agent. In embodiments in which the anti-BL binder also hasprotein A binding affinity, such as when the anti-BL binder is rabbitantibody, the two control zone capture agents may become linked. Inaddition, either or both the protein A and anti-BL binder can beimmobilized on the control zone using a carrier protein such as BSA.

In another embodiment, one control zone capture agent can be attached atthe control zone and another control zone capture agent can be appliedlater for example by flowing to the control zone with the sample. Forexample, when the binders are a cloxacillin binder and a BL binder, thecontrol zone can include protein A and anti-BL binder. Alternatively,the control zone can include protein A and the anti-BL binder can bebound to the BL binder and added to the strip with the BL binder such ason the sample pad. BL binder will retain beta-lactam binding activityafter binding to anti-BL binder, but not after binding to a beta-lactamdrug in the sample. In such an embodiment, the control zone captureagent is selected for its affinity to the anti-BL binder at a site notoccupied by BL binder. Examples of such capture agents include ProteinA, protein G, recombinant protein AG or other substances such assubstances that bind, for example, to the constant region of anantibody.

In an embodiment, detectably labeled multianalyte binder (“labeledmultianalyte binder) and detectably labeled specific binder (“labeledspecific binder”) are combined with unlabeled binders, such as unlabeledantibodies. The unlabeled antibodies are selected for their affinity toantibiotics to which the other binders, for example the multianalytebinder, are oversensitive. Such unlabeled binders compete with labeledmultianalyte binders for a specific antibiotic and, thereby, reduce testsensitivity to the antibiotic/analyte to which the unlabeled bindershave affinity. The unlabeled binders can have affinity for some or allof the analyte to which the labeled multianalyte binder has affinity. Byincluding unlabeled binders with affinity for some, but not all, of theanalytes to which a competing labeled binder has affinity, sensitivityof the test to those selected antibiotics/analytes will be reduced.

In some embodiments there is excess capture reagent in the control zone.Excess capture reagent will provide consistent control zone detection.In other control zone embodiments there is an excess of the generalantibody binder, such as protein A, relative to the multianalyte bindercapture agent, such as anti-BL binder, such as rabbit antireceptor. In aparticular example using protein A and rabbit anti-BL binder, thecontrol zone will have sufficient excess protein A available forcapturing cloxacillin binder and binding the anti-BL binder.

In certain embodiments, in a negative sample the control zone willcapture fewer labels as compared to any one of the test zones, in apositive sample for a single analyte or family, the control zone willcapture more labels than one of the test zones and fewer labels than theother and, if positive for all analytes, each of the test zones willcapture fewer labels as compared to the control zone.

Including multiple capture agents at the control zone, such as bothprotein A, or other general antibody binder, and specific antibody to abinder, such as anti-BL binder, provides the possibility of a test stripfor detection of multiple analytes such as multiple antibiotics. Forexample, a test strip to detect sulfonamides, tetracyclines, amphenicolsor macrolides, possibly combined on a test strip to detect beta-lactams,with a single control zone for comparison to multiple test zones for thedifferent antibiotics. A control zone can have multiple specificantibodies to particular binders, multiple multianalyte binderantibodies, multiple antibody binding proteins or combinations thereof.Similarly, the control zone can include multiple antibodies of differenthost species and not necessarily include a general antibody binder. Forexample, if one binder is a mouse monoclonal antibody and another is arabbit polyclonal antibody, the control zone can include an anti-mouseantibody and an anti-rabbit antibody.

In another embodiment, the assay can be in the form of a so-calledsandwich assay using different types of binders, such as a combinationof polyclonal and monoclonal antibodies or a combination of differentspecies polyclonal antibodies. Tests to detect multiple analytes using asandwich assay can include a plurality of labeled analyte binders thatbind with different analytes. The test zone can include multipleimmobilized binders for the analyte, for example with affinity todifferent regions of the analyte to which the labeled antibody hasaffinity. The control zone could include a combination of immobilizedcapture agents including, for example, combinations of anti-speciesantibodies for reactions to each of the different analyte binders. Thecontrol zone could also combine, depending on the binders used, acombination of general antibody binding protein, such as protein A, andan antibody for one of the binders to which protein A does not haveadequate affinity, such as anti-mouse antibody. In such an embodiment,as with the other embodiments described herein in which the non-sandwichformat is used, either the analyte binder can be labeled with a visiblelabel or, in an embodiment, the label can require a further reactionsuch as when the label is an enzyme or substrate of an enzyme linkedimmunosorbent assay (ELISA).

In another embodiment, one type of binder, for example a labeledpolyclonal antibody, can be used to detect a single analyte or a groupof related analytes. One test zone and one control zone can be used tocapture the labeled binder. The control zone can include multiplecapture agents, each with different affinity to the binder, such as eachwith affinity to different parts of the binder. For example, if thelabeled antibody is a rabbit polyclonal, the control zone capture agentscan include, for example, an anti-rabbit antibody and an antibodybinding protein such as protein A. By including different control zonecapture agents, it may be possible to improve binder capture at thecontrol zone and, thereby, improve the control zone signal.

In some embodiments BL binder is isolated directly from, for example,B.st., by immobilized ligand affinity chromatography techniques that arewell known in the art.

The binder can also be expressed from other hosts by inserting into thehost genome the sequence of the BL binder from B.st.

Another example of a useful multianalyte binder, that can be used incertain embodiments, includes a beta-lactam binding protein isolatedfrom Bacillus lichenformis. Other possibly useful binders includemacromolecules, such as monoclonal or polyclonal antibodies, hormonereceptors and enzymes and synthetic receptors such as those generatedthrough molecular imprinting of synthetic polymers or molecularimprinting inside dendrimers.

EXAMPLES

One embodiment includes a three-zone test for detection of penicillin-G,amoxicillin, ampicillin, cloxacillin, ceftiofur and cephapirin at safelevel. This embodiment includes two test zones and one control zone. Thefirst test zone solution includes a cloxacillin-BSA conjugate at 0.1-2mg/ml, buffered with 10 millimolar (mM) sodium phosphate, pH 5.5-7.0,containing 20% sucrose. The mixture was sprayed 1.6 cm above the bottomedge of a nitrocellulose membrane at a rate of 0.6 μl/cm. The secondtest zone solution includes a ceforanide-BSA conjugate at 0.55-0.8 mg/mlin 10 mM sodium phosphate, pH 6.9, containing 15% sucrose sprayed ontonitrocellulose using a BIODOT® sprayer. The second test zone was sprayed2.2 cm above the bottom edge of the nitrocellulose at a rate of 0.8μl/L.

The ceforanide-BSA conjugate, was made by using the amino group on theceforanide to add a sulfhydryl group. Next a cross-linking agent, forexample,Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC), was added to link the sulfhydryl group on the ceforanidederivative to the amino group on the carrier protein.

The cloxacillin-BSA conjugate was made by activating the carboxyl groupof cloxacillin with a carbodiimide and N-hydroxysulfosuccinimide (S-NHS)and subsequently binding the cloxacillin activated compound to aminogroups on BSA. Cloxacillin (1 g) is dissolved in dry DMSO. The S-NHS wasdissolved in dry DMSO and added to the cloxacillin solution. To thissolution (approximately 30 ml), 0.5 ml of diisolpropyl carbodiimide wasimmediately added and the reaction allowed to proceed for 2 to 3 hours.The reaction was then extracted 3 times with dry petroleum ether and theDMSO portion was retained. The activated cloxacillin in DMSO wasgradually added to a solution of BSA (4-g/70 ml) in 0.02 M sodiumphosphate buffer at pH 7.2. This reaction was allowed to proceedovernight with mixing. The resulting cloxacillin-BSA conjugate wasdesalted to remove free cloxacillin.

In an alternative method for making the cloxacillin-BSA conjugate,dissolve 1 gram of sulfhydryl blocked BSA in 9 ml of 0.1 M potassiumcarbonate buffer at pH 10.0. While stirring add dropwise a cloxacillinsolution (100 mg/ml in DMSO) to the BSA solution. Place in a 15 mlcentrifuge tube and mix overnight in an orbital shaker. Neutralize thesolution by adding 4.0 ml of 0.4 M phosphate buffer, pH 6.3. Desalt thereaction on FPLC using one high prep 26/10 desalting column equilibratedwith 0.02 M phosphate buffer, pH 7.2. To give a theoretical substitutionof moles of cloxacillin per mole of BSA of 5.0, 32.5 mg of cloxacillinor 0.325 ml is added to the BSA solution.

In this embodiment the gold label includes a combination of BL bindergold conjugate and monoclonal cloxacillin antibody (cloxacillin binder)gold conjugate. Approximately 30% of the solution includes gold coatedwith 600 U (units) of beta-lactam binder (one unit is defined as theamount of purified antibiotic binder able to bind 1000 counts per minuteof radiolabeled antibiotic, such as when using the Charm II System)purified from B.st. and approximately 20% consists of gold coated with1000 U of purified cloxacillin binder. To the remainder of the solutionwas added a 10 mM sodium phosphate buffer, pH 7.4, containing 40%sucrose and 10% BSA. The combined bead solution was sprayed at 0.7 to0.9 μl/cm with 2 to 4 passes onto POREX®.

The monoclonal antibody to cloxacillin was purified by ammonium sulfateprecipitation at 50% saturation and dialyzed against 20 mM sodiumphosphate buffer, pH 7.2, containing 50 mM sodium chloride and thenpurified using a protein A column.

The BL binder was purified by passing a solubilized receptor solutionthrough a column containing a bound beta-lactam ligand possessingbeta-lactam binding activity. As the impure receptor solution was passedthrough the column, the receptor was bound to the beta-lactam andretained on the column. The column was washed to further removeimpurities and then an elution solution was used, containinghydroxyl/amine, to breaks the bond between the receptor and thebeta-lactam allowing the receptor to be eluted from the column.

The control zone was made as follows:

a) Preparation of BSA-Protein A Conjugate

1) 8 bottles of 5 mg recombinant protein A was dissolved in about 12 mlof 0.16 M borate buffer, pH 8.1. Next, 13 mg S-SMCC was weighed out anddissolved in 1 ml of DMSO. A 30 μl portion of the S-SMCC solution wasadded to the protein A solution and mixed for 1 hour. 500 mg BSA wasthen dissolved in 8 ml of 0.4 M phosphate buffer, pH 6.3. The Protein Aand BSA solutions were mixed together and brought to pH 7.0 withadditional 0.4 M phosphate buffer. The solution was then brought to 30ml with water.

b) Preparation of Rabbit Anti-Receptor Antibody.

The antibody was made in rabbits using purified beta-lactam receptorfrom Geobacillus stearothermophilus as the immunogen. The rabbitanti-receptor antibody was purified on a protein A column and diluted to5 mg/ml protein for storage.

c) Application to Test Strip

BSA-Protein A conjugate prepared as described above was diluted 25 foldin 10 mM phosphate buffer, pH 6.95, containing 5% sucrose. Rabbitanti-receptor was prepared as described above and diluted 150 times in10 mM phosphate buffer, pH 6.95, containing 25% sucrose. The two dilutesolutions were then combined and immobilized onto the nitrocellulose onthe control zone by spraying the solution onto the control zone.

With reference to FIG. 1 and the above specific example, the sampleapplication area 7 includes the cloxacillin binder gold conjugate andthe BL binder gold conjugate. Test zone 9 can include the immobilizedcloxacillin-BSA conjugate and test zone 4 can include the immobilizedceforanide-BSA conjugate. The control zone 5 could, therefore, includethe BSA-protein A conjugate and rabbit anti-receptor antibody.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the test strip apparatus 8, comprised of nitrocellulosemembrane 3 and a binder application area 7 onto which the labeledbinders can be added. The binder application area can be, for example,POREX® attached to solid support 2. If a sample pad 1 is used, sample iscontacted to sample pad 1. Alternatively, sample can be applied directlyto the binder application area 7. If a sample pad 7 is used, sampleflows from sample pad 1 to the sample application area 7 containing, inan example, labeled specific binder and labeled multianalyte binder.Labeled specific binder and labeled multianalyte binder will bindanalyte from the sample and flow along the nitrocellulose membrane 3 totest zone 9 and test zone 4. A portion of labeled specific binder andlabeled multianalyte binder, unbound by sample analyte, will be capturedat the related test zone. Remaining labeled binder, whether or not boundby sample analyte, will flow to, and can bind to, control zone 5. Somelabeled binder may also flow past the control zone and into the disposalpad 6. A stronger signal in the control zone as compared to the testzone is a positive result. A weaker signal in the control zone ascompared to the test zone is a negative result.

FIG. 2 shows the test strip components prior to application of samplesolution. The arrows indicate the direction of sample flow from thebinder application area 7. Not shown is a sample pad 1 or other testsolution application area that may precede the binder application area7. The binders in the application area are, in this example, labeledspecific binder 21, in this example labeled cloxacillin binder, andlabeled multianalyte binder 22, in this example labeled BL binder. Thecloxacillin binder 21 includes cloxacillin binding site 31, detectablelabel 32, for example gold particle, and protein A binding site 33. TheBL binder 22 includes beta-lactam binding site 34, anti-BL binderbinding site 35 and label 32. The test zone 9 includes immobilizedcloxacillin 38. In this embodiment, the beta-lactam ring of theimmobilized cloxacillin is opened, depicted in this drawing as a break24 in the ring, to reduce or eliminate the affinity of the beta-lactambinding site 34 of the BL binder 22. Binding site 39 on immobilizedcloxacillin 38 is available to capture specific binder 21 unbound bycloxacillin from the sample. The test zone 4 includes immobilizedrepresentative beta-lactam 25 such as ceforanide. BL binder binding site37 is available to capture BL binder 22 unbound by beta-lactam from thesample. The control zone 5 includes protein A 41. The control zone 5also includes anti-BL binder 42. Protein A can capture cloxacillinbinder 21 through attachment to the protein A site 33. As a result,protein A can capture both bound and unbound cloxacillin binder. Ifmixed together, Protein A 41 can also bind to anti-BL binder 42 at thecontrol zone 5 to form an anti-BL binder-protein A complex 43 that mayretain the ability to capture both cloxacillin binder 21 and BL binder22. Anti-BL binder 42 can capture BL binder 22 unbound by beta-lactamfrom the sample or BL binder 22 bound by beta-lactam from the sample.

FIG. 3 shows a sample that is negative for both beta-lactams generallyand the specific beta-lactam cloxacillin at the appropriate level ofdetection. After application of the sample, test components flow out ofbinder application area 7. Cloxacillin binder 21, unbound by cloxacillinfrom the sample, can be captured by cloxacillin immobilized on the firsttest zone 9 to form a complex 51 that can be detected. Similarly, BLbinder 22, unbound by beta-lactam from the test sample, can be capturedby beta-lactam 25 immobilized on the second test zone 4 to form complex52 that can be detected. Protein A 41 in control zone 5, whethercomplexed to anti-BL binder or alone, can capture both bound and unboundcloxacillin binder 21 that flow past the test zone 9 without beingcaptured. In this figure complex 45 includes cloxacillin binder unboundby cloxacillin from the sample and captured at the control zone. Theanti-BL binder 42, whether complexed to protein A, or alone, can captureboth bound or unbound BL binder that flow past the test zone 4 withoutbeing captured, for example to form complex 44 in the control zone 5. Inthis figure some sample beta-lactam-BL binder complex 55 and samplecloxacillin-cloxacillin binder complex 54 are not captured and insteadflow to the disposal pad 6. Not all labeled binder, whether or not boundby analyte from the sample, is necessarily captured at the test zones orcontrol zone. Remaining label can flow to the disposal zone 6. Thenegative result can be determined, in the drawing, by counting thelabels 32 in the control zone and test zones. In this example, thecontrol zone 5 has two labels. The test zone 4 has four labels and thetest zone 9 has four labels. Since the control zone has fewer labelsthan either of the test zones the test is negative for both cloxacillinand other beta-lactams. Although one beta-lactam from the sample wascaptured by BL binder 22 to form a complex that was captured 44 at thecontrol zone 5, and one each of BL-binder and cloxacillin binder werebound by antibiotic to form complex 55 and complex 54 respectively, thatflowed to the disposal zone 6, the semi-quantitative nature of the testis reflected in the negative result.

FIG. 4 shows a test that is positive for both cloxacillin specificallyand beta-lactams. Three cloxacillin binders 21 are bound at test zone 9,two BL binders 22 are bound at test zone 4 and five binders are bound atcontrol zone 5. Control zone 5 includes captured BL binder-label-samplebeta-lactam complex 44, captured sample cloxacillinbinder-label-cloxacillin complex 47 and captured unbound labeledcloxacillin binder complex 45. Disposal zone 6 includes cloxacillinbinder 21 and BL binder-sample beta lactam complex 55, both of whichslipped through uncaptured.

FIG. 5 shows a test that is positive for beta-lactams and negative forcloxacillin specifically. Three labels are captured at the control zone5 and two labels are captured at test zone 4 while four labels arecaptured at test zone 9. Three labels are not captured and flow to thedisposal zone 6.

FIG. 6 shows a test that is negative for beta-lactams and positive forcloxacillin specifically. Two labels are captured at the test zone 9 andfour labels are captured at the test zone 4 as compared to three labelscaptured at the control zone 5. Three labels are not captured and flowto the disposal zone 6.

It should be noted that the figures, particularly FIGS. 2-6, are highlysimplified depictions designed to exemplify both the various mechanismsof capture, binding and affinity and test result interpretation.Although in these figures the mechanisms are largely described relativeto detection of beta-lactams, including cloxacillin, the methods andassays described herein are not so limited.

1. A lateral flow device for detecting a plurality of analytes in a testsolution, the test solution including a sample and more than one binder,the binder configured for generating a detectable signal and capable ofcombining with an analyte present in the sample to form a binder-analytecomplex, the device comprising: a test strip configured to allow thetest solution to flow, the test strip comprising: (i) more than one testzone, each test zone having immobilized thereon a test zone captureagent that can capture at least one of the binders that has not formed abinder-analyte complex; and (ii) a single control zone having aplurality of control zone capture agents combined together within thecontrol zone, each of the plurality of control zone capture agentshaving different binding affinities for the binders and each able tocapture at least one of the binders, whether or not the binder hasformed a binder-analyte complex, wherein capture of any of the bindersat any of the test zones and the control zone results in a detectablesignal and wherein a greater signal in the control zone as compared toany one of the test zones indicates a positive result.
 2. The device ofclaim 1 wherein the control zone capture agents have binding affinity toeach other.
 3. The device of claim 1 wherein at least one of the controlzone capture agents comprises an antibody and another of the controlzone capture agents comprises an antibody binding protein.
 4. The deviceof claim 1 wherein at least one of the binders comprises a labeledmultianalyte binder disposed on the test strip.
 5. The device of claim 4wherein the multianalyte binder comprises a labeled beta-lactam binder.6. The device of claim 1 further comprising a labeled specific binderdisposed on the test strip.
 7. The device of claim 6 wherein the labeledspecific binder comprises a labeled cloxicillin binder.
 8. The device ofclaim 1 wherein at least one of the control zone capture agentscomprises an anti-beta-lactam binder.
 9. The device of claim 1 furthercomprising at least one unlabeled binder disposed on the test stripwherein the unlabeled binder reduces test sensitivity relative to theanalyte to which the unlabeled binder has affinity.
 10. The device ofclaim 1 wherein at least two of the control zone capture agents areantibodies.
 11. The device of claim 1 wherein at least one of thecontrol zone capture agents comprises an antispecies antibody.
 12. Thedevice of claim 1 wherein at least one of the control zone captureagents comprises an antibody binding protein.
 13. The device of claim 12wherein the antibody binding protein comprises protein A.
 14. The deviceof claim 1 wherein at least one of the control zone capture agents isimmobilized on the control zone through attachment to a carrier protein.15. The device of claim 1 wherein the carrier protein comprises bovineserum albumin.
 16. The device of claim 1 wherein one of the plurality ofcontrol zone capture agents comprises an antibody binding protein andanother of the plurality of control zone capture agents comprises anantibody to which the antibody binding protein has affinity, and whereinthe antibody is applied to the test strip with the binders, and flows,either with the binders or attached to one or more of the binders, tothe control zone.
 17. The device of claim 1 wherein one of the binderscomprises a labeled beta-lactam binder and wherein one of the controlzone capture agents comprises an anti-beta lactam binder.
 18. The deviceof claim 1 wherein one of the control zone capture agents comprises apolyclonal antibody and another comprises a monoclonal antibody.
 19. Thedevice of claim 1 wherein each of the binders comprises an antibody froma different species of animal and at least one of the control zonecapture agents comprises an antibody to one of those different species.20. The device of claim 1 wherein the test solution further comprisesadditional reagents that are reconstituted when the sample is added tothe test strip.
 21. The device of claim 1 comprising two bindersdisposed on the test strip wherein one of the binders comprises alabeled beta lactam binder and the other of the two binders comprises alabeled cloxicillin binder.